1. Technical Field
The field of the invention relates to fluorescence polarization assays for detecting HDC activity that can be used in the diagnosis of disease and identification of HDC inhibitor agents.
2. Background Information
Histamine is a potent bioamine with multiple activities in various pathological and physiological conditions (Jutel M, Watanabe T, Akdis M, Blaser K, Akdis C A: Immune regulation by histamine. Curr. Opin. Immunol 2002; 14:735-740). In addition to its well-characterized effects on the acute inflammatory and allergic responses, histamine regulates several aspects of antigen-specific immune responses (Schneider E, Rolli-Derkinderen M, Arock M, Dy M: Trends in histamine research: new functions during immune responses and hematopoiesis. Trends Immunol 2002; 23:255-263). Recent findings, such as the discovery of a novel histamine receptor (H4) on immunocompetent cells and the demonstration of a role for H1 and H2 receptors on T helper cell polarization, have generated much interest in the immune-regulatory mechanisms triggered by histamine (Schneider E, Rolli-Derkinderen M, Arock M, Dy M.; Trends Immunol. 2002 May; 23(5):255-63).
Histidine decarboxylase (HDC) is the rate-limiting enzyme in the biosynthesis of histamine (Watanabe T, Yamatodani A, Maeyama K, Wada H: Pharmacology of α-fluoromethylhistidine, a specific inhibitor of histidine decarboxylase. Trends Pharmaceutical Sci 1990; 11:363-367.) Mammalian HDC is a member of a large family of pyridoxal 5-phosphate (PLP)-dependent enzymes (Christen P, Mehta P: From Cofactor to enzymes. The molecular evolution of pyridoxal-5′-phosphate-dependent enzymes. Chemical Record 2001; 1:436-447.) HDC is expressed in most tissues but the highest levels are found in the skin, the GI track and the airways. HDC is a 74 Kd enzyme that is converted to a shorter 54 Kd form (Yatsunami K, Tsuchikawa M, Kamada M, Hori K, Higuchi T: Comparative studies of human recombinant 74- and 54-kDa L-histidine decarboxylase. J. Biol. Chem. 1995; 270:30813-30817). Both forms are active in vitro but they are not found in the same subcellular compartments; the 74 Kd form being found predominantly in the endoplasmic reticulum (Tanaka S, Nemoto K, Yamamura E, Ohmura S, Ichikawa A: Degradation of the 74 kDa form of 1-histidine decarboxylase via the ubiquitin-proteasome pathway in a rat basophilic/mast cell line (RBL-2H3). FEBS Letters 1997; 417:203-207).
The recent generation of HDC-deficient mice provided a good system to study the role of endogenous histamine in a broad range of normal and disease processes (Ohtsu H, Watanabe T: New functions of histamine found in histidine decarboxylase gene knockout mice. Biochem Biophys Res Commun 2003; 443-447). The HDC−/− mice have a reduced number of mast cells and reduced granular content such as mast cell proteases (Ohtsu H, Tanaka S, Terui T, Hori Y, Makabe-Kobayashi Y, Pejler G, Tchougounova E, Hellman L, Gertsenstein M, Hirasawa N, Sakurai E, Buzas E, Kovacs P, Csaba G, Kittel A, Okada M, Hara M, Mar L, Numayama-Tsuruta K, Ishigaki-Suzuki S, Ohuchi K, Ichikawa A, Falus A, Watanabe T, Nagy A: Mice lacking histidine decarboxylase exhibit abnormal mast cells. FEBS 2001; 502:53-56.) These mice show reduced airway hyperresponsiveness (Kozma G T, Losonczy G, Keszei M, Komlosi Z, Buzas E, Pallinger E, Appel J, Szabo T, Magyar P, Falus A, Szalai C: Histamine deficiency in gene-targeted mice strongly reduces antigen-induced airway hyper-responsiveness, eosinophilia and allergen-specific IgE. International Immunol. 2003; 15:963-973, reduced vascular permeability (Ohtsu et al. Plasma extravasation induced by dietary supplemented histamine in histamine-free mice. Eur J. Immunol. 2002; 32:1698-708), reduced skin inflammation (Ghosh A K, Hirasawa N, Ohtsu H, Watanabe T, Ohuchi K: Defective angiogenesis in the inflammatory granulation tissue in histidine decarboxylase-deficient mice but not in mast cell-deficient mice. J. Exp. Med. 2002; 195:973-982.) and increased bone density (Fitzpatrick L A, Buzas E, Gagne T J, Nagy A, Horvath C, Ferencz V, Mester A, Kari B, Ruan M, Falus A, Barsony J. Targeted deletion of histidine decarboxylase gene in mice increases bone formation and protects against ovariectomy-induced bone loss. Proc Natl Acad Sci USA. 2003; 100(10):6027-32). Thus, potent inhibitors of HDC activity might prove useful in allergic, inflammatory, immunological, bone and cardiovascular disorders. Histamine has also been shown to be a positive regulator of proliferation in some types of cancers (Hegyesi H, Somlai B, Varga V L, Toth G, Kovacs P, Molnar E L, Laszlo V, Karpati S, Rivera E, Falus A, Darvas Z. Suppression of melanoma cell proliferation by histidine decarboxylase specific antisense oligonucleotides. J Invest Dermatol. 2001 July; 117(1):151-3).
The biological role of histamine has been extensively studied with pharmacological approaches using histamine receptor specific agonists or antagonists. Despite the important role of HDC in allergic and inflammatory responses, very few small molecule inhibitors of this enzyme are known. Most of these inhibitors were discovered by rational design strategies and are histidine analogues. A well characterized HDC inhibitor is the irreversible inhibitor alpha-fluoromethyl histidine (Watanabe T, Yamatodani A, Maeyama K, Wada H. Pharmacology of alpha-fluoromethylhistidine, a specific inhibitor of histidine decarboxylase. Trends Pharmacol Sci. 1990 11:363-7).
The ability to identify novel classes of HDC inhibitors is limited by the lack of assays that are suitable to HTS. The most commonly used assay to measure HDC activity is based on the o-phthalaldehyde (OPT) method (Roskoski R, Roskoski L M: A rapid histidine decarboxylase assay. Analytical Biochem. 1978; 87:293-297.) This assay is not selective for histamine over histidine and involves a chromatographic separation of the enzyme product from the substrate. Another more sensitive HDC assay utilizes the conversion of [14C]-labeled histidine to [14C]-labeled-histamine. Thin layer chromatography is then used to resolve substrate and product. Histamine ELISA kits could potentially be adapted to measure HDC activity. However, these assays require an acetylation step (acetylated histamine) to reach any useful selectivity and sensitivity. Moreover, these procedures require many washing steps rendering them less amenable for HTS.